Project Summary/Abstract Clostridium difficile infection (CDI) is the leading cause of antibiotic-associated colitis and is responsible for significant morbidity, mortality and increased healthcare costs. Despite the significance of CDI, there are major gaps in our understanding of the pathogenesis of this infection. Antibiotics disrupt the indigenous gut microbiota, reducing resistance to C. difficile colonization. However, our knowledge of how the gut microbiota confers resistance to CDI is rudimentary, presenting a significant roadblock to improving preventative and therapeutic approaches against this infection. My long-term goal is to understand how the gastrointestinal tract microbiota mediates colonization resistance against C. difficile. The overall objective of this application is to define metabolites associated with changes in the gut microbiota that contribute to C. difficile colonization and pathogenesis. Using an untargeted metabolomics approach, we have shown that the intestinal environment of antibiotic-treated mice was characterized by major shifts in metabolic profiles. Following antibiotic administration, we detected increases in primary bile acids, carbohydrates, and amino acids and decreased free fatty acids, secondary bile acids and dipeptides; reflecting the diminished metabolic activity of the gut microbiome. Subsequently, we demonstrated that C. difficile could utilize many of these metabolites for in vitro germination and growth. The central hypothesis is that the availability of specific nutrients that support C. difficile growth in the gut after antibiotic treatment is responsible for the observed decrease in colonization resistance. The rationale for the proposed research is that understanding the role the gastrointestinal metabolome plays in C. difficile pathogenesis has the potential to improve preventative and therapeutic approaches for this infection. Guided by strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Identify metabolites in the gastrointestinal tract that contribute to C. difficile colonization and pathogenesis; and 2) Determine the physiological concentrations of gut metabolites that modulate C. difficile pathogenesis. Under the first specific aim, we will use an untargeted metabolomics approach to identify candidate biomarkers from the murine gastrointestinal tract prior to CDI and during different stages of infection. Under the second specific aim, we will use a targeted metabolomics approach to confirm and quantitate metabolites that were significantly affected in specific aim 1, prior to CDI and during different stages of infection. We will also use in vitro studies to confirm their role in C. difficile germination, growth and toxin production. The approach is innovative, because we are using new mass spectrometry technology in a different way, to help solve important biological questions that will improve public health. The proposed research is significant, because it will lead to the identification of novel biomarkers and potential targets for therapeutic interventions to prevent or treat CDI. My overall career goal is to establish an independent research career bridging the field of metabolomics and biomedical infectious diseases, with emphasis on understanding Clostridium difficile pathogenesis. My long-term research interests have always included studying the impact of disease and how it impacts human health. With the advent of omics technologies complex communities, including the gastrointestinal tract, can be defined. The expertise and co-mentorship of both Dr. Vincent Young and Dr. Charles Burant ensures success of this research project and my continued success a research scientist. The combined resources that my mentors and collaborators share will allow me access to mouse models of C. difficile infection and the Metabolomics Core Facility, which includes access to state of the art mass spectrometry equipment and trained experts in metabolomics. Finally, these studies will provide me with the opportunity to learn the methodologies related to the emerging field of metabolomic profiling, including the design of studies, sample preparation, metabolite extraction and analysis by the latest mass spectrometer based methods and the processes of data analysis and bioinformatics interpretation of the acquired data. The mentorship plan detailed in this proposal and further didactic coursework in ethics, bioinformatics, statistics, and workshops on metabolomics will help me to become an independent researcher in the field of biomedical infectious diseases and metabolomics.